Reticulocytes are immature red blood cells (or RBCs) containing residual cytoplasmic ribosomes which are formed when developing red blood cells extrude their nuclei into the bone marrow under normal hemopoietic conditions. These cells spend two to three days in bone marrow prior to being released into the peripheral blood circulation and one to two days in circulation before losing their ribonucleic acid (RNA) content and reaching maturity. The reticulocyte population in the peripheral blood is approximately 1% of the total erythrocyte population in normal subjects, and the majority of the reticulocytes are of mature form. The hormone erythropoietin, which stimulates and regulates erythrocyte production, is responsible for the release of the reticulocytes from the bone marrow. Young reticulocytes contain larger quantities of RNA, and this characteristic can be employed to distinguish mature and immature reticulocytes.
Reticulocyte counting is a valuable blood test, and it is very widely done. The number of reticulocytes in peripheral blood circulation is used to study and evaluate erythropoietic activity of bone marrow. Subjects with suppressed bone marrow activity, e.g., those patients undergoing chemotherapy, exhibit a lower reticulocyte percentage and a higher mature reticulocyte population. Conversely, those patients with stimulated bone marrow function, such as individuals with hemolytic anemia, show higher reticulocyte percentage and a lower mature reticulocyte population than those observed in normal and healthy subjects.
Reticulocyte identification and enumeration is based on the finding of residual ribosomes and nucleic acid in early nonnucleated red blood cells. This diagnostic test has long been done manually by staining and precipitating residual ribonucleic acid with dyes such as new methylene blue and brilliant cresyl blue. The precipitated RNA will appear either as cytoplasmic dots or filaments under microscope. Unfortunately, the manual reticulocyte test has long been known for its poor accuracy and precision. Irregular staining and random distribution of reticulocytes on the blood film, variations in the preparation of the stained film and the stain used, presence of artifacts resulting from precipitation of free and unbound dye, poor microscope focus, and differences in definition of the reticulocytes are some of the factors that can introduce error or contribute to the inaccuracy of manual reticulocyte counting. However, another problem with manual techniques is that suitable, long-lasting, highly accurate reference controls are often lacking.
In recent years, automated reticulocyte counting has been possible due to advances in flow cytometry. Flow cytometric analysis of reticulocytes depends on the binding of suitable fluorescent dyes to residual ribosomes and nucleic acid. Excitation of the stained cells with a laser beam and detection of forward scatter and side fluorescence provides information on size and nucleic acid content of red blood cells. Acridine orange, auramine O, pyronine Y, thioflavin T, and propidium iodide have been used in flow cytometric reticulocyte counting methods. These dyes provide sufficient resolution between reticulocytes and background erythrocytes for on-line instrumental reticulocyte analyses.
Because of these newer flow cytometric techniques, higher degrees of precision have been obtained for instrumental reticulocyte analysis. One reason for the better precision of the automated analysis is that many more cells can be counted per analysis than only the roughly 1000 cells that are counted in the manual analysis. Also, the instrumental method of flow cytometry involves setting the threshold to exclude mature red blood cells which is an objective method as compared to the manual method in which the identification of reticulocytes is a subjective decision.
However, the flow cytometry procedures currently used also rely on reference controls that suffer from various inaccuracies and which cannot be used with manual analyses. In particular, the reticulocyte reference controls used in these procedures commonly involve the binding of various fluorochromes to RBCs or polymer particles which produces certain inaccurracies and which results in a Gaussian reticulocyte distribution on the flow cytometers.
At present, there is thus an absence of a highly accurate reticulocyte reference control which can be used in assessment of bone marrow activity and in maximizing quality control with regard to cytometric procedures involving blood cells. Further, there is a lack of adequate reference control materials which are highly reliable, long-lasting and which can be used in both manual and flow cytometric techniques used to monitor a variety of diseases and therapeutic treatments involving blood cells. It is thus highly desirable to obtain a suitable reference control by which reticulocyte count can be accurately determined, and it is also desirable that such a reference control be suitable for use with both manual and flow cytometers.
It is presently known to employ a reversible osmotic lysis procedure in order to encapsulate various compounds into erythrocytes. In such a procedure, red blood cells are hypotonically dialyzed in the presence of the desired compound, such as enzyme, so that the erythrocytes swell up and form pores of sufficient size so that the desired compound enters the cells. The treatment with hypotonic solution is followed by hypertonic dialysis to reseal the pores formed on the cell membrane so as to encapsulate or entrap the desired compound inside the cells. This process is disclosed in a variety of patents and other references, including, e.g., U.S. Pat. Nos. 4,931,276 and 4,652,449, Am. J. Vet. Res. 44:1159 (1983), Am. J. Vet. Res. 42:667 (1981), Toxicol. App. Pharm. 83:101 (1986) and Biochim. Biophys. Acta 496:136 (1977), all incorporated herein by reference.
Although these loaded erythrocytes have been used in specific biotherapeutic applications such as enzyme therapy and other methods wherein specific compounds are introduced into the blood stream (see, e.g., J. Lab. Clin. Med. 96:307 (1980), incorporated herein by reference), the use of such loaded erythrocytes as reference controls for cytometric procedures has not previously been disclosed or suggested. It has recently been discovered by the inventors that such loaded erythrocytes can be successfully used to prepare a reliable, stable and accurate reference control that can be used in both manual and flow cytometric techniques such as the counting of reticulocytes.